We investigated the effects of interleukin (IL)-10 administration on allergen-induced Th2 cytokine production, eosinophilic inflammation, and airway reactivity. Mice were sensitized by intraperitoneal injection of ragweed (RW) adsorbed to Alum and challenged by intratracheal instillation of the allergen. Sensitization and challenge with RW increased concentrations of IL-10 in bronchoalveolar lavage (BAL) fluid from undetectable levels to 60 pg/ml over 72 h. Intratracheal instillation of 25 ng of recombinant murine IL-10 at the time of RW challenge further elevated BAL fluid IL-10 concentration to 440 pg/ml but decreased BAL fluid IL-4, IL-5, and interferon-gamma levels by 40-85% and eosinophil numbers by 70% (P < 0.0001). Unexpectedly, the same IL-10 treatment increased airway reactivity to methacholine in spontaneously breathing mice that had been sensitized and challenged with RW (P < 0.001). IL-10 treatment in naive animals or RW-sensitized mice challenged with PBS failed to increase airway reactivity, demonstrating that IL-10 induces an increase in airway reactivity only when it is administered in conjunction with allergic sensitization and challenge. The results demonstrate that IL-10 reduces Th2 cytokine levels and eosinophilic inflammation but augments airway hyperreactivity. Thus, despite its potent anti-inflammatory activity, IL-10 could contribute to the decline in pulmonary function observed in asthma.

Epidemiological studies have linked ambient particulate matter (PM) levels to an increased incidence of adverse cardiovascular events. Yet little is definitively known about the mechanisms accounting for the cardiovascular events associated with PM exposure. The goal of this study was to determine the effects of ultrafine (<0.1 microm) PM exposure on ischemia-reperfusion (I/R) injury. ICR mice were exposed to 100 microg of PM or vehicle by intratracheal instillation. Twenty-four hours later, mice were anesthetized with pentobarbital sodium (60 mg/kg), the left anterior descending coronary artery was ligated for 20 min, flow was restored for 2 h, and the resulting myocardial infarct (MI) size was evaluated. PM exposure doubled the relative size of the MI compared with the vehicle control. No difference was observed in the percentage of the left ventricle at risk for ischemia. PM exposure increased the level of oxidative stress in the myocardium after I/R. The density of neutrophils in the reperfused myocardium was increased by PM exposure, but differences in the number of blood leukocytes, expression of adhesion molecules on circulating neutrophils, and activation state of circulating neutrophils 24 h after PM exposure could not be correlated to the increased I/R injury observed. Additionally, aortas isolated from PM-exposed animals and studied in vitro exhibited a reduced endothelium-dependent relaxation response to acetylcholine. These results indicate that exposure to ultrafine PM increases oxidative stress in the myocardium, alters vascular reactivity, and augments injury after I/R in a murine model.

Intratracheal administration of interleukin-10 (IL-10) has been reported to inhibit allergic inflammation but augment airway hyperresponsiveness (AHR). In the present study, airway and smooth muscle responsiveness to methacholine (MCh) were compared in wild-type (WT) and IL-10-deficient (IL-10-KO) mice to investigate the role of endogenous IL-10 in AHR development. Naive WT and IL-10-KO mice exhibited similar dose-dependent increases in airway resistance (Raw) to intravenous MCh. Sensitization and challenge with ragweed (RW) induced a twofold increase in responsiveness to intravenous MCh in WT mice, but hyperresponsiveness was not observed in similarly treated IL-10-KO mice. Likewise, tracheal rings from RW-sensitized and -challenged WT mice exhibited a fourfold greater responsiveness to MCh than IL-10-KO tracheal preparations. Measurements of airway constriction by whole body plethysmography further supported the Raw and tracheal ring data (i.e., AHR was not observed in the absence of IL-10). Interestingly, factors previously implicated in the development of AHR, including IL-4, IL-5, IL-13, IgA, IgG1, IgE, eosinophilia, and lymphocyte recruitment to the airways, were upregulated in the IL-10-KO mice. Treatment with recombinant murine IL-10 at the time of allergen challenge reduced the magnitude of inflammation but reinstated AHR development in IL-10-KO mice. Adoptive transfer of mononuclear splenocytes to IL-10-sufficient severe combined immunodeficient mice indicated that lymphocytes were an important source of the IL-10 impacting AHR development. These results provide evidence that IL-10 expression promotes the development of allergen-induced smooth muscle hyperresponsiveness.

Recent observations demonstrated that translation of mRNAs may occur in axonal processes at sites that are long distances away from the neuronal perikaria. While axonal protein synthesis has been documented in several studies, the mechanism of its regulation remains unclear. The aim of this study was to investigate whether RNA interference (RNAi) may be one of the pathways that control local protein synthesis in axons. Here we show that sciatic nerve contains Argonaute2 nuclease, fragile X mental retardation protein, p100 nuclease, and Gemin3 helicase-components of the RNA-induced silencing complex (RISC). Application of short-interfering RNAs against neuronal beta-tubulin to the sciatic nerve initiated RISC formation, causing a decrease in levels of neuronal beta-tubulin III mRNA and corresponding protein, as well as a significant reduction in retrograde labeling of lumbar motor neurons. Our observations indicate that RNAi is functional in peripheral mammalian axons and is independent from the neuronal cell body or Schwann cells. We introduce a concept of local regulation of axonal translation via RNAi.

Treatment of mice with heat-killed (HK)Mycobacterium

Animal models exhibiting high homology with humans at the genetic and pathophysiological levels will facilitate identification and validation of gene targets underlying asthma. In the present study, a nonhuman primate model of allergic asthma was developed by sensitizing cynomolgus monkeys to dust mite antigen. Sensitization elevated allergen-specific serum IgE and IgG levels, and peripheral blood mononuclear cells isolated from sensitized animals released IL-4, IL-5, and IL-10, but not IFN-gamma. Aerosolized allergen decreased dynamic compliance and induced airway inflammation and hyperresponsiveness to aerosolized histamine. Albuterol and dexamethasone inhibited the airway constriction and allergen-induced inflammation, respectively. Airway wall remodeling that included goblet cell hyperplasia, basement membrane thickening, and smooth muscle hypertrophy was particularly evident in neonatally sensitized animals. In contrast to animals sensitized as adults, neonatally sensitized animals exhibited increased sensitivity to adenosine and larger allergen-induced changes in airway resistance and dynamic compliance. These results demonstrate that sensitization of cynomolgus monkeys with dust mite induces asthmalike symptoms, some of which may be dependent on age at the time of sensitization.

GAL-021 behaved as a breathing control modulator in rodents and nonhuman primates and diminished opioid-induced respiratory depression without compromising opioid analgesia. It acted predominantly at the carotid body, in part by inhibiting KCa1.1 channels. Its preclinical profile qualified the compound to enter clinical trials to assess effects on breathing control disorders such as drug (opioid)-induced respiratory depression and sleep apnea.

Epidemiological studies have linked levels of particulate matter (PM) in ambient air to cardiovascular mortality and hospitalizations for myocardial infarction (MI) and stroke. Thrombus formation plays a primary role in potentiating acute cardiovascular events, and this study was undertaken to determine whether pulmonary exposure to PM alters hemostasis. PM was collected from the Chapel Hill, NC airshed and was administered to mice by intratracheal instillation at a dose previously shown to exacerbate myocardial ischemia-reperfusion injury. Twenty-four hours after exposure, an increase occurred in the number of circulating platelets and plasma concentrations of fibrinogen and soluble P-selectin. The concentration of tissue factor pathway inhibitor (TFPI) in plasma was decreased, whereas the plasma concentration of plasminogen activator inhibitor (PAI-1) was increased. Consistent with these observations, bleeding time from a tail-tip transection was shortened. These results provide evidence that PM exposure alters hemostasis in otherwise healthy animals and may thereby promote clot formation and impede clot resolution in susceptible individuals. The results also establish definite hemostatic endpoints that can be used to further investigate the effects of dose and particle characteristics on the toxicity of ambient particles.